The proportional bias of four methods frequently used for determining low concentrations of protein was evaluated with human protein fractions (Cohn Fractions II, III, IV and V). Each fraction was assigned a protein concentration value which was determined by the biuret method using purified human serum albumin monomer to establish the calibration. All four methods (Coomassie blue dye-binding, Lowry et al., ultraviolet absorption, and the immunoephelometric) gave acceptable results for fraction V (albumin). The ultraviolet absorption and the Lowry methods overestimated the three globulin fractions (II, III, and IV) and the Coomassie blue dye-binding and the immunoephelometric methods underestimated these globulin fractions. The least proportional bias for the globulin fractions was with the Lowry method.